Insights Into an Unexplored Component of the Mosquito Repeatome: Distribution and Variability of Viral Sequences Integrated Into the Genome of the Arboviral Vector Aedes albopictus

Abstract

The Asian tiger mosquito Aedes albopictus is an invasive mosquito and a competent vector for public-health relevant arboviruses such as Chikungunya ( Alphavirus ), Dengue and Zika ( Flavivirus ) viruses. Unexpectedly, the sequencing of the genome of this mosquito revealed an unusually high number of integrated sequences with similarities to non-retroviral RNA viruses of the Flavivirus and Rhabdovirus genera. These Non-retroviral Integrated RNA Virus Sequences (NIRVS) are enriched in piRNA clusters and coding sequences and have been proposed to constitute novel mosquito immune factors. However, given the abundance of NIRVS and their variable viral origin, their relative biological roles remain unexplored. Here we used an analytical approach that intersects computational, evolutionary and molecular methods to study the genomic landscape of mosquito NIRVS. We demonstrate that NIRVS are differentially distributed across mosquito genomes, with a core set of seemingly the oldest integrations with similarity to Rhabdoviruses . Additionally, we compare the polymorphisms of NIRVS with respect to that of fast and slow-evolving genes within the Ae. albopictus genome. Overall, NIRVS appear to be less polymorphic than slow-evolving genes, with differences depending on whether they occur in intergenic regions or in piRNA clusters. Finally, two NIRVS that map within the coding sequences of genes annotated as Rhabdovirus RNA-dependent RNA polymerase and the nucleocapsid-encoding gene, respectively, are highly polymorphic and are expressed, suggesting exaptation possibly to enhance the mosquito’s antiviral responses. These results greatly advance our understanding of the complexity of the mosquito repeatome and the biology of viral integrations in mosquito genomes.

Improved reference genome of the arboviral vectorAedes albopictus

“…We searched AalbF2 for nix and located it in an approximately 917 kb scaffold (NW_021838423.1). The nix sequence is male-specific as indicated by the Chromosome Quotient analysis (44) using Illumina reads obtained from male and female mosquitoes of the Foshan strain (45). A part of the nix gene was previously identified in Ae.…”

Immunity to infections in arboviral vectors by integrated viral sequences: an evolutionary perspective

“…To gain insights into nrEVE evolution, we analyzed patterns of occurrence of viral integrations in the geographic samples and their polymorphism in relation to slow-and fast-evolving Ae. aegypti genes (Pischedda et al, 2019). We reasoned that, if viral integrations result from fortuitous events and are viral fossils, their distribution should be governed by drift and their polymorphism should evolve at a neutral rate (Aswad and Katzourakis, 2012;Katzourakis, 2013).…”

“…We calculated the G12 and G1/2 statistics genome-wide and verified whether To further confirm that some nrEVEs are adaptive, we compared their polymorphism with that of two previously validated sets of fast-evolving and conserved Ae. aegypti genes, respectively (Pischedda et al, 2019). If nrEVEs are biological inactive relics and have reached fixation by genetic drift, they should accumulate mutations at the host mutation rate.…”

“…The presence or absence of each viral integration characterized from the Ae. aegypti reference genome (AaegL5 assembly) was analyzed in genomic resequencing of individual mosquitoes using an inhouse bioinformatic pipeline, which also allows for sequence polymorphism analysis (Pischedda et al, 2019). The pipeline allows to detect the presence of nrEVEs in each tested individual, but not to distinguish between heterozygote and homozygote status.…”

“…Because of the stringency used in the call for nrEVE presence (i.e. minimum five reads with at least 30 consecutive nucleotides with mapping quality higher than 20) (Pischedda et al, 2019), a short sequence that shares sequence identity with a longer one could be erroneously called absent in all individuals tested because of reads shared with the longer nrEVE. For this reason, a total of 29 viral integrations, which were called absent in all individuals, were excluded from further analyses (supplementary table 1).…”